DNA And RNA Assembly Nanotechnology Based On Small Circular DNA Molecules

Since Watson and Crick found the principle of base paring and double helix structure of DNA, molecular biology has developed rapidly. Take advantages of its minuscule size and’stiffness’ structure, large information storage and loyal base pairing, DNA as a Molecular building block has been attracted tremendous attention to build functional nanomaterial and motif. Professor Nadrian C. Seeman from New York University, considered to be the founder of the field of DNA nanotechnology, proposed the possibility of using DNA as a structural material for the assembly of geometrically defined objects with nanoscale features for bottom-up self-assembly in1982. Since that time, a set of addressable DNA nanostructures have been fabricated into one-(ID), two-(2D), and three-dimensions (3D) in the past thirty years, and applications in nanoelectronics, biosensing and computation have been developed."DNA origami" technology was introduced by Rothemund in2006, in his study; a long single-stranded DNA strand (scaffold) was folded into a series of desired2D shapes with the help of hundreds of short oligonucleotides, called staple strands.RNA/DNA hybrid origami technology just appeared in2013which imitated the DNA origami technology. In the procedure, the traditional DNA scaffold was replaced by a single long strand RNA which was produced from in vitro transcription. Then, the RNA scaffold was folded by the short synthetic DNA strands into predesigned structures according to the base pairing principles. RNA/DNA hybrid origami nanostructures have potentials to expand the variety of structural components for the design and construction of novel nanostructures.At present, DNA nanotubes can potentially template the growth of nanowires, act as molecular motors and drug delivery vehicles. In particular, drug delivery vehicles based on DNA are a promising class of biodegradable and biocompatible drug Nanocarriers. Their sizes, shapes, and targeting ligand presentation on their surfaces are more precisely tunable than any of the currently available Nanomaterials. All in all, DNA Nanotubes are an important component class in Nanomachine.This thesis describes the method to produce periodical long single-stranded RNA molecular via the classic amplification method named RNA-Rolling circle transcription (RCT). Imitate the DNA origami techniques, the periodical long single-stranded RNA molecular was folded into predesigned stable RNA/DNA nanowires. Inspired by this work, cyclic DNA molecules which used as template for transcription are exploited as new motif to build micron size DNA Nanotubes. The specific research works are as follows:1. Researches on RNA/DNA hybridization origami technology. Firstly, a mild and effective amplification method named Rolling circle transcription (RCT) has been adopted which was developed on Rolling circle amplification (RCA). And the periodical long single-stranded RNA scaffold was produced with the help of T7RNAP and rNTP mixture incubated2-4hours at37℃. According to the principles of DNA origami, we first designed three different kinds of Nanowires. The successful formations of the Nanowires were confirmed by atomic force microscopy (AFM) imaging. Experiment results were highly consistent with the theoretical value. In order to verify the feasibility of this method, another96-base circular sequence was adopted to as template to produce another periodical long single-stranded RNA molecule which would be act as scaffold in the following experiments. We designed another kind of Nanowire. Via AFM imaging, we observed the Nanowires clearly. And the height, the width and the length agreed with the theoretical value too.2. Researches on dense DNA Nanotubes formation by using small circular DNA strands (<100nt) as new building blocks. With the help of T4Ligase and a splint strand (usually20bp), the straight DNA strand which was phosphorylated at the5end can be closed to form a circular DNA strand. The circular DNA strands were bundled together via the formation of crossovers. We chose two different circular strands-96nt sequence and64nt-sequence.Crossovers can be formed twice or thrice between the adjacent circular DNA strands, respectively. And we also do research on the effect of placing T at the circular DNA strands where the crossovers were formed. The Experiment result indicated that adding Ts were not essential for the formation of this kind of Nanotubes.